The overall goal of this proposal is to identify and characterize cellular proteins that bind to viral hemorrhagic fever viruses. Specifically, we focus on RNA viruses from four families: the Arenaviruses Junin virus and Lassa Fever virus; the Bunyavirus Crimean-Congo hemorrhagic fever virus; the filoviruses Ebolavirus and Marburg virus; and the Flavivirus dengue virus. These emerging viruses pose significant risks to human health and national security. While each differs in the types of proteins encoded, they all share similar disease outcomes, causing severe, often fatal infections in humans; most are classified as NIAID Category A Priority Pathogens. However, due to the high level containment needed to work with many of them, there is a significant gap in our understanding of these viruses and no specific therapies are currently available. Like all viruses, these emerging viruses rely on host cell proteins in order to replicate. This dependence represents a potential Achilles heel in the virus life cycle that may be exploited to develop new approaches to treat viral infections. With the exception of dengue virus, however, only a small number of virus-host cell protein interactions have been reported for the viruses in this study, and no systematic analysis of emerging virus-host cell protein interactions has been performed. In this project we develop and evaluate high quality maps of emerging virus-human protein interactions. In Aim 1 we use complementary yeast two-hybrid screening and co-affinity purification plus mass spectrometry approaches to identify cellular proteins that bind to emerging viruses. To facilitate comparisons of the virus-host cell interactions of the diverse viruses in this study, all human proteins identified in the yeast two-hybrid assays will be tested for interactions with all virus proteins. During the course of work we develop innovative techniques to further increase the capacity of yeast two-hybrid screening. In aim 2 we evaluate the significance of the virus-human protein interactions by searching for enriched annotation features among the cellular binding partners, integrating the interaction network with cellular pathways and existing virus-host cell interaction data, and identifying small molecule inhibitors of the cellular proteins in the network. In aim 3, subset of high-interest cellular targets will be experimentally interrogated for their contributionto virus infection by using RNA interference to and small molecule inhibitors to inhibit the expression or activity of the cellular binding partner. Cellular proteins required for virus replication will be further examined with state-of-the-art molecular techniques to determine the mechanisms by which they contribute to virus replication The data generated from this project will improve our understanding of the functions of individual viral proteins, provide insight into the overall strategies used by viral hemorrhagic fever virus to interface with their host cells, an may lead to the discovery of new targets for treatments of emerging viruses.